LAMP combined with Pyrococcus furiosus Argonaute for the ultrasensitive and highly specific point-of-care test platform for Listeria monocytogenes detection

Abstract

Listeria monocytogenes is an important foodborne pathogen that causes serious infectious diseases in humans and animals. Rapid, accurate, and sensitive detection in the field is critical for the timely implementation of control measures and ensuring food safety. The study established a method that combines loop-mediated isothermal amplification (LAMP) technology and Pyrococcus furiosus Argonaute ( Pf Ago) reporter system, which can detect L. monocytogenes hly gene levels as low as 1.8 × 10 1 copies. To overcome the demands on instrumentation and equipment, lateral flow strip (LFS) technology was combined for the first time in a LAMP- Pf Ago-based system to detect L. monocytogenes . The method was highly sensitive, with a limit of detection of 1.8 × 10 2 copies, and specificity results showed no cross-reactivity with other pathogens. The performance of the method was comparable to that of real-time fluorescence quantitative PCR (qPCR) in the detection of 18 food origin samples. Moreover, the LAMP- Pf Ago reaction was equipped a low-cost thin metal bath as a simple amplification device, allowing it to detect L. monocytogenes samples in shorter time, highlighting its potential as a portable, rapid, accurate, and visual nucleic acid assay. The method can facilitate the early detection of pathogenic infections and promoting food safety detection technologies. Schematic diagram of Pf Ago-based Listeria monocytogenes assay workflow. • Lateral flow strip readout was introduced for the first time in the LAMP- Pf Ago system to detect L.monocytogenes . • The LAMP- Pf Ago assay detects DNA at the L. monocytogenes hly gene levels in about a short period of time. • The ultrasensitive and highly specific point-of-care test platform allows visualization without equipment.

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